1. Aligning Sequences You have learned about: Data & databases Tools
Amino Acids
Protein Structure
Today we will discuss: Aligning sequences
After this, you are ready to carry out
a bioinformatics research project!
©CMBI 2009

2. TRANSFER OF INFORMATION!
Why align sequences?
The problem: There a lots of sequences with unknown structure and/or function There are a few sequences with known structure and/or function
Alignment can help:
If sequences align well, they are likely to be similar
If they are similar, then they very likely share structural and/or functional aspects
If one of them has known structure/function, then alignment gives us insight in structural and/or functional aspects of the aligned sequence(s)
TRANSFER OF INFORMATION!
©CMBI 2009

3. Sequence Alignment (1)
A sequence alignment is a representation of a whole series of evolutionary events, which left traces in the sequences.
Things that are more likely to happen during evolution should be most prominently observed in your alignment.
The purpose of a sequence alignment is to line up all residues in the sequence that were derived from the same residue position in the ancestral gene or protein.
©CMBI 2009

4. gap = insertion or deletion
Sequence Alignment (2) A B
gap = insertion or deletion A B
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5. Structural alignment
To carry over information from a well studied protein sequence and its structure to a newly discovered protein sequence, we need a sequence alignment that represents the protein structures today, a structural alignment.
The implicit meaning of placing amino acid residues below each other in the same column of a protein (multiple) sequence alignment is that they are at the equivalent position in the 3D structures of the corresponding proteins!!
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6. Examples
1) the 3 active site residues H, D, S, of the serine protease we saw earlier
2) Cysteine bridges (disulfide bridges): STCTKGALKLPVCRK
TSCTEG--RLPGCKR
©CMBI 2009

7. Transfer of information
Such information can be:
Phosphorylation sites
Glycosylation sites
Stabilizing mutations
Membrane anchors
Ion binding sites
Ligand binding residues
Cellular localization
Typically what one finds in the feature (FT) records of Swissprot!
©CMBI 2009

8. Significance of alignment
One can only transfer information if the similarity is significantly high between the two sequences.
Schneider (group of Sander) determined the “threshold curve” for transferring structural information from one known protein structure to another protein sequence: If the sequences are > 80 aa long, then >25% sequence identity is enough to reliably transfer structural information. If the sequences are smaller in length, a higher percentage of identity is needed.
Structure is much more conserved than sequence!
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9. Significance of alignment (2)
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10. Aligning sequences by hand
Most information that enters the alignment procedure comes from the physico-chemical properties of the amino acids.
Examples: which is the better alignment (left or right)?
1) CPISRTWASIFRCW CPISRTWASIFRCW
CPISRT---LFRCW CPISRTL---FRCW
2) CPISRTRASEFRCW CPISRTRASEFRCW
CPISRTK---FRCW CPISRT---KFRCW
©CMBI 2009

11. Aligning sequences by hand (2)
Procedure of aligning depends on information available:
Use “only” identity of amino acid and its physico-chemical properties. This is more or less what alignment programs do.
Also use explicitly the secondary structure preference of the amino acids. Example: aligning 2 helices when sequence identity is low
Use 3D information if one or more of the structures in the alignment are known.
In most cases you will start with a alignment program (e.g. CLUSTAL) and then use your knowledge of the amino acids to improve the alignment, for instance by correcting the position of gaps.
©CMBI 2009

12. Helix
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13. Positional preferences in helices (1)
total
H H H H H
ASP
Dataset of good helices from PDB files
Count all Asp residues in & before helices
Identify preferential positions for Asp residues
Position 1 in helix
©CMBI 2009

14. Positional preferences in helices (2)
Fill this table for all 20 amino acids
Use this information when aligning helices who have low percentage of sequence identity
total
H H H H H
ALA
CYS
ASP
GLU
(…)
TRP
TYR
Position 1 in helix
©CMBI 2009

15. Aligning 2 helices when sequence identity is low
Helix 1:
S G V S P D Q L A A L K L I L E L A L K
Helix 2:
G T S L E T A L L M Q I A Q K L I A G
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16. Aligning 2 helices when sequence identity is low (2)
S G V S P D Q L A A L K L I L E L A L K
5 5
G T S L E T A L L M Q I A Q K L I A G 5
Final alignment: S G V S P D Q L A A L K L I L E L A L K
- G T S L E T A L L M Q I A Q K L I A G
©CMBI 2009

17. Use of 3D structure info (1)2
If you know that in structure 1 the Ala is pointing outside and the Ser is pointing inside: Where does the Arg in structure 2 go? (and what will CLUSTAL choose?)
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18. Use of 3D structure info (2)
A ILE CYS ARG LEU PRO GLY SER ALA GLU ALA VAL
B1 VAL CYS ARG THR PRO GLU ALA ILE
B2 VAL CYS ARG THR PRO GLU ALA ILE
©CMBI 2009

19. An even more real example
A ILE CYS ARG LEU PRO GLY SER ALA GLU ALA VAL
B1 VAL CYS ARG THR PRO GLU ALA ILE
B2 VAL CYS ARG THR PRO GLU ALA ILE
G- -
PP-
S-T
LT-
A-P
RRR
EEE
CCC
IVV
AAA
V I I
©CMBI 2009

20. We have seen that alignments ….
Are crucial for being able to transfer information
Can be optimized by using secondary structure preferences (e.g. helix positioning)
Can be optimized by using 3D structure info
©CMBI 2009

21. Multiple sequence alignments
If we have more than two sequences aligned, the alignment is called a multiple sequence alignment (MSA)
MSA’s can:
confirm or improve pair-wise sequence alignments
reveal structural information (e.g. cys-bridges)
validate PROSITE search results
©CMBI 2009

22. MSA for improvement of pair-wise alignments
CWPVAASYGR
CWPT---YGR
CWPTA-SYGR
CWPTLGLFGR
©CMBI 2009

23. MSA and cysteine bridges
Multiple sequence alignments can reveal structural information:
ASCTRGCIKLPTCKKMGRCTGY
STCTKGALKLPVCRKMGKSSAY
ATSTHGCMKLPCSRRFGKCSSY
TSCTEGCLRLPGCKRFGRCTSY
TTCTKGLLKLPGCKRFGKSSAY
ASSTKGCMKLPVSRRFGRCTAY
©CMBI 2009

24. MSA to validate PROSITE results (1)
PROSITE glycosylation pattern:
N-{P}-[ST]-{P} where N is the glycosylation site.
PROSITE Syntax: A-[BC]-X-D(2,5)-{EFG}-H
Means: A
B or C
Anything
2-5 D’s
Not E,F or G H
©CMBI 2009

25. MSA to validate PROSITE results (2)
The chance of finding N-{P}-[ST]-{P} is rather high.
So how can you be sure? Look at the multiple sequence alignment:
ASLRNASTVVTIGDTITGNLTLASYHW
GSIKNGSSVITLPGTMEGNLSTTTYHY
ATLRNASTVMEINGTITGDLTLASFHW
©CMBI 2009

26. What you have learned today (and will need for your own project)
A good sequence alignment is necessary to carrying over information between proteins.
Putting amino acids below each other in a sequence alignment implies that you predict that they are on equivalent positions in both proteins.
If the aligned sequences are > 80 aa long, then >25% sequence identity is enough to reliably transfer structural information.
You need to use all structural information available to you to optimize the sequence alignment. This can be real 3D data, but can also be “just” your own knowledge about the properties and preferences of the amino acids.
©CMBI 2009